1. Field of the Invention
This invention relates to an improved method for in vitro protein synthesis from a substantially prokaryotic and eukaryotic cell-free extract.
It also relates to an improved method of assaying such synthesis.
It also relates to a kit which includes a vial containing substantially prokaryotic and eukaryotic cell-free extract, a vial containing a translation cocktail, and a vial containing a control messenger ribonucleic acid (mRNA).
2. Description of the Prior Art
The conversion of amino acids into protein involves a complicated process involving a number of different components. Genetic information is carried within a cell by a substance known as deoxyribonucleic acid (DNA). DNA is a nucleic acid consisting of different nucleic acids arranged in a double helix chain with the order of attachment determining the genetic code the DNA contains. In a eukaroytic cell, the DNA is contained within the visibly evident nucleus contained within such a cell. In a prokaryotic cell, the DNA is scattered about within the entire cell since such a cell does not have a true nucleus. By a process called transcription, the DNA causes the formation of messenger ribonucleic acid (mRNA), an RNA fraction of intermediate molecular weight which transmits information from DNA to the protein-forming system of the cell. In the case of a eukaryotic cell, this involves the migration of the mRNA through the wall of the nucleus into the cytoplasm of the cell. The mRNA so produced is then picked up by a ribosome, a ribonucleoprotein particle found in the cytoplasm, which converts the message received from the mRNA into a specific protein by a process known as translation. Another component involved in translation is transfer ribonucleic acid (tRNA), an RNA fraction of low molecular weight, existing in 20 species, each of which combines with one amino acid species, transferring it within the cytoplasm to the ribosome. The message from the DNA is contained in mRNA in units known as codons which are three nucleic acid segments which request a particular amino acid depending upon their sequence. The ribosome reads the codon of the mRNA and selects the appropriate tRNA by matching that codon with a tRNA having the complementary anti-condon. The particular amino acid carried by that tRNA is then attached by the ribosome to the end of the growing peptide chain. Thus by sequentially reading the mRNA chain, the ribosome forms a chain of amino acids into a complete protein.
In studying the process by which proteins are formed by living organisms it was determined that research would be aided if the aforesaid protein synthesis could be performed in vitro.
Such methods of synthesizing proteins in vitro are well known in the prior art. For example, see Weissback, H. and Ochoa, S., Ann. Rev. Biochem., 45, 191 (1976); Lucas-Lenard, J. and Lipmann, F., Ann. Rev. Biochem., 40, 409 (1971); Haselkorn, R., and Rothman-Denes, L. B., Ann. Rev. Biochem., 42, 397 (1973); Lucas-Lenard, J. and Beres, L., the Enzymes, 10, 53 (1974); and Ochoa, S. and Mazumder, R., the Enzymes, 10, 1 (1974), all incorporated herein by reference.
In principle, protein synthesis can be regulated at many different levels; from preferential replication of the gene and transcription of the gene into RNA to processing RNA and final protein synthesis. Much remains to be learned about the agents and mechanisms which might be involved in protein synthesis.
One area of protein synthesis which has received a great deal of attention has been translation. There has been a great deal of discussion as to whether different cells differ in their ability to translate different mRNAs. It has been found, in general, that mRNAs from one cell type can be translated efficiently in cell extracts, or intact cells, of a different type without definite requirements for specific factors or other components.
Numerous eukaryotic and prokaryotic cell-free systems efficiently (and faithfully) translate messenger RNAs from viral or eukaryotic origin. In vitro translation systems have been reported for Krebs II ascites tumor, rat and mouse liver, HeLa cells, mouse L cells, Chinese hamster ovary (CHO) cells, other mammalian cells, reticulocytes, wheat germ, rye embryo and other sources. These systems can all be used in assaying RNA molecules for messenger activity. The main disadvantage to the study of mechanisms of translation in vitro is the fact that some of the extracts show high levels of endogenous protein synthesizing activity, i.e. experimental mRNA added to such systems for study must compete with the pre-existing (endogenous) mRNA of the cells from which the extract was produced.
The preparation of an extract efficient in translating in vitro exogenously added mRNA from a wide variety of cells is a potentially useful tool in the study of specificity and control of translation. For example, the rabbit reticulocyte lysate system is reported to contain all factors required for the translation of any eukaryotic mRNA. A reticulocyte is a cell in an intermediate stage in the production of red blood cells from bone marrow cells. Because it has already extruded its nucleus and thus its DNA, it causes less competition from endogenous mRNA produced by the DNA when assaying exogenous mRNA. Unfractionated reticulocyte lysate preparations can be used in prokaryotic and eukaryotic cell-free protein synthesis systems. However, such preparations contain already produced endogenous mRNA, and added mRNA can only be translated to the extent that it can compete with the endogenous mRNA. Treatment of reticulocyte preparations with the enzyme micrococcal nuclease effectively inactivates endogenous mRNA, yet retains activity of other components required for protein synthesis. An example of such a treatment is described in Pelham, H. R. B. and Jackson, R. J., "An Efficient mRNA-Dependent Translation System from Reticulocyte Lysates", Eur. J. Biochem., 67, 247 (1967), incorporated herein by reference. Using this in vitro mRNA-dependent translation system, the mechanisms of protein synthesis, the screening of recombinant DNA and the locating of protein coding regions within eukaryotic or viral DNA can be studied.
Once such prokaryotic and eukaryotic cell-free extract had been prepared, it was combined with the desired experimental exogenous mRNA, tracer and translation cocktail and mixed throughly. The tracer was any of the amino acids expected to be incorporated into the protein by the translation process which was labelled in a suitable manner, e.g. by radioactivity, so that its percentage of incorporation into the protein produced could be determined. The translation cocktail typically consisted of the various components to promote the translation process such as a stabilizer for the tracer, buffer to maintain the system at basically neutral pH, the biochemical energy supply required for the translation process, and amino acids expected to be employed in the translation process other than that employed as the tracer. This mixture was then allowed to incubate for a sufficient period of time, followed by separation of the labelled protein from the remainder of the solution by standard methods well known in the art, such as precipitation using trichloroacetic acid, etc. This separation was followed by methods of analysis well known in the prior art, such as polyacrylamide gel electrophoresis, immunoprecipitation, etc.
A number of problems exist in the prior art. For instance, in preparing the translation cocktail, the prior art suggests that all the amino acids present in the protein to be synthesized, other than the one used as the tracer, should be added to the translation cocktail. Preparing such a cocktail is an onerous, expensive, and time-consuming task. Furthermore, once such a cocktail is prepared, it is unusable with tracers other than the one for which it is prepared.
Another problem is the lack of any standard of comparison, when reviewing protein synthesis of another researcher. To make such a comparison necessitates the complete reproduction of the reported research results.
Another disadvantage concerns the lack of any means for determining where a mistake had been made, such as an improperly prepared reagent, etc., when the desired protein is not produced by the exogenous mRNA. The only means for checking a new protein synthesis is to synthesize a known mRNA and repeat the entire experimental procedure to determine whether the exogenous mRNA is defective or whether a mistake in technique or reagents is involved. Synthesizing or isolating a known mRNA and repeating the experimental procedure can take up to several days.
Accordingly, there is a need for a system providing a convenient reference standard for comparing the research of different investigators. Furthermore, there is a need for a system allowing various tracers to be incorporated without requiring the reformulation of the translation cocktail. In addition, a need also exists for a convenient and readily available model system for use in trouble-shooting an experimental system when problems arise.